Pkcδ-Mediated SGLT1 Upregulation Confers the Acquired Resistance of NSCLC to EGFR Tkis

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ARTICLE OPEN PKCδ-mediated SGLT1 upregulation confers the acquired resistance of NSCLC to EGFR TKIs

Chia-Hung Chen1,2,3,19, Bo-Wei Wang4,5,19, Yu-Chun Hsiao4,5,6,19, Chun-Yi Wu7, Fang-Ju Cheng 6,8,9, Te-Chun Hsia1,3,10, Chih-Yi Chen11, 12,13 14 4,6 2,8 15,16,17 4 4,9 Yihua Wang ✉, Zhang Weihua , Ruey-Hwang✉ Chou , Chih-Hsin Tang ✉ , Yun-Ju Chen , Ya-Ling Wei , Jennifer L. Hsu , Chih-Yen Tu 1,2 , Mien-Chie Hung 4,5,6 and Wei-Chien Huang 4,5,6,18

The tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) have been widely used for non-small cell lung cancer (NSCLC) patients, but the development of acquired resistance remains a therapeutic hurdle. The reduction of glucose uptake has been implicated in the anti-tumor activity of EGFR TKIs. In this study, the upregulation of the active sodium/glucose cotransporter 1 (SGLT1) was found to confer the development of acquired EGFR TKI resistance and was correlated with the poorer clinical outcome of the NSCLC patients who received EGFR TKI treatment. Blockade of SGLT1 overcame this resistance in vitro and in vivo by reducing glucose uptake in NSCLC cells. Mechanistically, SGLT1 protein was stabilized through the interaction with PKCδ- phosphorylated (Thr678) EGFR in the TKI-resistant cells. Our ﬁndings revealed that PKCδ/EGFR axis-dependent SGLT1 upregulation was a critical mechanism underlying the acquired resistance to EGFR TKIs. We suggest co-targeting PKCδ/SGLT1 as a potential strategy to improve the therapeutic efﬁcacy of EGFR TKIs in NSCLC patients.

Oncogene (2021) 40:4796–4808; https://doi.org/10.1038/s41388-021-01889-0

INTRODUCTION have adenocarcinoma histology. Importantly, the high prevalence The epidermal growth factor receptor (EGFR), a membrane-bound of activating EGFR mutations, including the exon 19 (L746-A750) tyrosine kinase receptor, has been found to be a critical oncogene deletion and L858R point mutation, were found in these patients in promoting the tumorigenesis, mitogenesis, and tumor progres- [5]. These activating mutations alter the protein structure of EGFR sion of various cancer types, including non-small cell lung cancer at its ATP-binding site and increase the binding affinity and (NSCLC) [1, 2]. Overexpression or somatic mutation of EGFR causes vulnerability to TKIs [6]. However, a frequent substitution of the aberrant activation of its tyrosine kinase and the dysregulation threonine residue to methionine at codon 790 (T790M) of EGFR of its downstream signals that contribute to the tumor growth exon 20 has been found to reduce the binding affinity with and progression and the poor prognosis of NSCLC patients [3]. gefitinib or erlotinib and thereby contributes to the development EGFR is therefore a rational and feasible therapeutic target for this of acquired resistance [1]. The second-generation irreversible TKI, disease. afatinib (BIBW2992, Gilotrif), was developed to target the activating Small molecule tyrosine kinase inhibitors (TKIs) targeting the EGFR mutant with less likelihood of the EGFR T790M point ATP-binding pocket of the EGFR kinase domain were developed mutation [7]. The third-generation TKI, osimertinib (AZD9291, and approved for NSCLCs [4]. Gefitinib (ZD1839, Iressa) and Tagrisso), was further designed to target the EGFR T790M mutation erlotinib (OSI-774, Tarceva) are two approved first-generation EGFR [8, 9]. The secondary EGFR mutations [10] and activations of TKIs for NSCLC. However, these drugs fail to achieve maximum alternative RTKs creating a bypass track [11, 12] have been therapeutic efficacy, with response rates of typically 10–20% across proposed as reasons for the failed responses to EGFR TKIs, yet fail a variety of malignancies [4]. Gefitinib and erlotinib particularly to fully account for the development of acquired resistance to benefit Asian NSCLC patients who are female, never-smokers, and these drugs in NSCLC patients.

1Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan. 2School of Medicine, China Medical University, Taichung, Taiwan. 3Department of Respiratory Therapy, China Medical University, Taichung, Taiwan. 4Center for Molecular Medicine, Research Center for Cancer Biology, and Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan. 5Drug Development Center, China Medical University, Taichung, Taiwan. 6The Ph.D. Program for Cancer Biology and Drug Discovery, China Medical University and Academia Sinica, Taichung, Taiwan. 7Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan. 8Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan. 9Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 10Department of Internal Medicine, Hyperbaric Oxygen Therapy Center, China Medical University Hospital, Taichung, Taiwan. 11Division of Thoracic Surgery, Department of Surgery, Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan. 12Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, Southampton, UK. 13Institute for Life Sciences, University of Southampton, Southampton, UK. 14Department of Biology and Biochemistry, University of Houston, Houston, TX, USA. 15Department of Medical Research, E-Da Hospital, Kaohsiung, Taiwan. 16School of Medicine for International Students, I-Shou University, Kaohsiung, Taiwan. 17Department of Pharmacy, E-Da Hospital, Kaohsiung, Taiwan. 18Department of Medical Laboratory Science and ✉ Biotechnology, Asia University, Taichung, Taiwan. 19These authors contributed equally: Chia-Hung Chen, Bo-Wei Wang, Yu-Chun Hsiao. email: [email protected] ; [email protected]; [email protected] Received: 5 August 2020 Revised: 18 May 2021 Accepted: 4 June 2021 Published online: 21 June 2021 C.-H. Chen et al. 4797 Several studies have shown that, in addition to delivering the corresponding ER clones, was dramatically and dose-dependently classic proliferation and survival signals, activated EGFR also decreased by reducing glucose concentrations from 4.5 to 0.1 g/L facilitates glucose utility and metabolic pathways through the in the culture medium in WST1 (Fig. 1a and Supplementary Fig. stabilization of glucose transporters and dysregulation of glyco- S1d) and cell counting assays (Fig. 1b). Similarly, ER clones of H322 lytic enzymes hexokinases and pyruvate kinase M2 (PKM2) that and HCC827 cells were also more tolerant to glucose deprivation promote tumor growth, epithelial-mesenchymal transition (EMT), in clonogenic formation assays (Fig. 1c). Flow cytometric analysis cancer stemness, and even immune evasion [13–17]. Recently, further showed that glucose deprivation increased the sub-G1 tyrosine kinase activation of EGFR was shown to enhance the population of parental H322 and HCC827 cells, but not their activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase- corresponding ER clones (Fig. 1d). 3 (PFKFB3), an essential glycolytic activator for the synthesis and Nutrition and energy deprivation trigger autophagy in a variety degradation of fructose-2,6-bisphosphate (F26BP) that contributes of cells [32], but the roles of autophagy in determining the to the survival of NSCLC cells [18]. EGFR signaling also links sensitivity to EGFR TKIs in NSCLC remain controversial [11, 33–36]. glycolysis with serine synthesis to support nucleotide biosynthesis Pretreatment of H322 and HCC827 cells with 3-methyladenine (3- and redox homeostasis [19]. Mounting evidence has proved that MA) and chloroquine (CQ), which suppress autophagic flux by EGFR impacts the rewiring of the glucose metabolic network to targeting PtdIns3Ks [37] and autophagosome-lysosome fusion [38] promote tumor progression in NSCLC [20]. respectively, prevented the suppression of viability induced by Clinically, the decrease in glucose metabolism measured by the glucose starvation (Supplementary Fig. S1e), suggesting that uptake of 2-[18F]-fluoro-2-deoxy-D-glucose ([18F]-FDG), the radi- autophagy contributes to glucose deprivation-induced cell death olabeled glucose analog, with positron emission tomography of these cell lines. In support of this finding, the reduction of (PET) analysis appears within days of initiating EGFR TKI therapy in glucose concentration induced the expression of autophagic NSCLC patients [21]. [18F]-FDG uptake is significantly decreased by marker (LC3β) and apoptotic markers (PARP or caspase 3 erlotinib treatment in sensitive, not insensitive, human NSCLC- cleavages) in H322 (Fig. 1e) and HCC827 (Fig. 1f) cells, but not xenograft mouse tumors [22, 23]. Changes in tumor glucose their corresponding ER clones. Consistently, the results from Cyto- metabolism precede decreases in tumor size in response to EGFR ID® Autophagy dye staining showed that glucose deprivation TKIs [22, 24]. These findings suggest that reduction of EGFR- enhanced the numbers (Fig. 1g) and levels (Fig. 1h) of mediated glycolysis may be involved in the anti-tumor activity of autophagosome-positive cells in the parental H322 cells, but not EGFR TKIs, and thus monitoring [18F]-FDG-PET uptake has been their ER clones. All of these findings indicated that ER NSCLC cells

1234567890();,: used to predict therapeutic responses to EGFR TKIs in lung cancer are more tolerant to glucose deprivation-induced autophagic patients [25–27]. Moreover, suppression of facilitative glucose cell death. transporter Glut was found to mediate the anti-cancer activity of TKIs in NSCLC cell lines bearing wild-type (wt) or mutant EGFR [28]. ER lung cancer cells exhibit higher glucose uptake through These studies suggested that the suppression of glucose uptake SGLT1 upregulation and metabolism may be essential to achieve the therapeutic Although TKI-resistant cells were more tolerant to glucose response of EGFR TKIs in NSCLC patients. However, elevated deprivation-induced cell death, inhibition of glycolysis with lactate secretion and glycolysis were found in long-term TKI hexokinase inhibitor 2-deoxy-D-glucose (2-DG) still suppressed treatment of NSCLC cells and in prostate cancer cells [29–31], the colony formation of the corresponding ER clones of H322 and raising the possibility that glucose metabolic re-wiring may HCC827 cells (Supplementary Fig. S2), suggesting that glucose contribute to the development of acquired resistance of NSCLC remains the critical energy source to generate ATP and cell to EGFR TKIs. building blocks through glycolysis for cell proliferation in these In this study, our data showed that NSCLC cells with acquired clones. The elevation in the extracellular acidification rate (ECAR), EGFR TKI resistance are more tolerant to low glucose-induced an indicator of glycolysis, in the corresponding ER clones was autophagy following a metabolic shift to higher glucose uptake higher than that in the parental H322 (Fig. 2a) and HCC827 and glycolysis activity due to upregulation of active glucose (Supplementary Fig. S3a) cells. By monitoring the engulfment of transporter SGLT1 by Thr678-phosphorylated EGFR, in a PKCδ- the fluorescent-labeled deoxyglucose analog 2-NBDG, the glucose dependent manner. Higher SGLT1 expression correlated with uptake ability of these cells was examined. The 2-NBDG uptake poorer clinical outcomes of EGFR TKI-treated NSCLC patients while was suppressed by erlotinib in H322 (Fig. 2b) and HCC827 targeting SGLT1 with pharmacological inhibitors suppressed (Supplementary Fig. S3b) cells but was higher in H322/ER and enhancements in glucose metabolism and lowered acquisition HCC827/ER clones in the presence of TKI. Interestingly, the ER#2 of EGFR TKI resistance in NSCLC xenograft-bearing mice. Our clone, but not its parental HCC827 cells, elicited higher ECAR results not only elucidate the metabolic mechanism underlying under the low (0.1 g/L) glucose condition (Supplementary Fig. the development of acquired resistance to EGFR TKIs but also S3c). Increases in 2-NBDG uptake in the corresponding ER clones indicate that targeting PKCδ/SGLT1 in combination with EGFR TKIs of H322 cells were more obvious under low glucose culture may benefit NSCLC patients. conditions than under normal glucose conditions (Fig. 2c). Active glucose transporters SGLTs are associated with 2-NBDG uptake in SGLT1-overexpressing HEK-293T cells (Supplementary Fig. S3d) RESULTS consistent with previous studies [39, 40], but compared with TKI-resistant cells are tolerant to autophagic cell death glucose, show a much lower binding affinity with 2-DG derivatives induced by glucose deprivation [41]. These findings imply the involvement of SGLTs in the higher To determine whether alternation of glucose metabolism plays a glucose uptake in these TKI-resistant clones. Indeed, the ability to role in the development of acquired resistance to EGFR TKIs, we increase the uptake of α-methyl-D-glucopyranoside (α-MDG), a first established erlotinib-resistant (ER) clones from wt EGFR- specific substrate for SGLTs, was apparent with H322/ER (Fig. 2d) expressing NSCLC lines NCI-H322 (H322) and NCI-H292 (H292) and and HCC827/ER (Supplementary Fig. S3e) cells compared with from an activating EGFR mutant-expressing HCC827 lung adeno- their parental cells. These findings support the contention that ER carcinoma cell line by culturing the cells in increasing concentra- cells express active glucose transporters that have a greater ability tions of erlotinib (by 2.5 µM every 2–3 weeks, up to a maintenance for glucose uptake. concentration of 10 µM for 3 months). Their resistance to erlotinib Among cancer-related glucose transporters [42, 43], only the was validated in WST-1 assays (Supplementary Fig. S1a–c). expression of SGLT1, not Glut1, Glut3, or SGLT2, was significantly Interestingly, the viability of the parental cell lines, but not their increased in different ER clones of H322 (Fig. 2e) and HCC827

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Fig. 1 The acquired erlotinib-resistant NSCLC cells were more tolerant to glucose deprivation. H322, HCC827, and their erlotinib-resistant (ER) cells were cultured in different concentrations of glucose. a–c The cell viability was measured in WST-1 (a), cell counting (b), and clonogenic (c) assays. d The changes in the sub-G1 population of the indicated cells in response to different concentrations of glucose were measured with PI staining in FACS analysis. e, f The levels of LC3β and caspase 3 cleavage in H322, HCC827 cells and their ER clones in response to glucose deprivation were analyzed by WB. g The low glucose concentration-induced autophagosome accumulation was detected by staining with Cyto-ID® Green autophagy dye in ﬂuorescence analysis was shown in the upper panel, and the number of autophagosome- positive cells was quantitated in the lower panel. h The effects of glucose deprivation on autophagosome formation in H322 and their ER cells were determined by FACS analysis (upper panel) and quantitated (lower panel). Data in (a), (b), (d), (g), and (h) represent mean ± sd from three independent experiments. *P < 0.05; ***P < 0.001 vs. control group, Student’s t-test. Data in (c), (e), and (f) are representative of three experiments.

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Fig. 2 The upregulated SGLT1 mediated the glucose uptake of the acquired erlotinib-resistant cells. a The changes in ECAR of H322 cells and their ER clones were measured by using the XF-24 Seahorse extracellular ﬂux analyzer. b 2-NBDG uptake ability of H322 cells and their ER clone was detected following EGFR-TKI-treatment. c, d 2-NBDG (c) and α-MDG (d) uptake ability of H322 cells and their ER clones were detected by FACS and Beckman LS6000 Scintillation Counter, respectively. e The protein levels of various glucose transporters in H322 cells and their ER clones were detected in WB with the indicated antibodies. f The effects of 100 μM phlorizin or 1 μM LX4211 on glucose uptake in the H322/ER#2 clone were measured under a low glucose concentration condition in a 2-NBDG assay. g The effects of SGLT1 shRNA on α-MDG uptake in the H322/ER#2 clone were measured under low glucose conditions by using the Beckman LS6000 Scintillation Counter. Data in (b), (c), (d), (f), and (g) represent mean ± sd from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. control, Student’s t-test. Data in (a) and (e) are representative of three experiments.

(Supplementary Fig. S3f) cells. In support of this ﬁnding, higher H322 (Fig. 3a) and HCC827 (Supplementary Fig. S4a) cells and their expression of SGLT1 in H292/ER clones than in parental cells viability under low glucose culture conditions (Fig. 3b and (Supplementary Fig. S3g) was also detected by the validated anti- Supplementary Fig. S4b). Consistently, shRNA-mediated depletion SGLT1 antibody (Supplementary Fig. S3h) in the xenograft tumor of SGLT1 limited the cell growth of different H322/ER (Fig. 3c) and tissues. These data suggest that increased levels of SGLT1 may HCC827/ER clones (Supplementary Fig. S4c), and re-sensitized enhance glucose uptake in ER cells. Indeed, treatments with SGLT these ER clones to cell death induced by glucose deprivation (Fig. inhibitors phlorizin and LX4211 (sotagliﬂozin, approved in the 3d and Supplementary Fig. S4d). Treatments with SGLT1 inhibitors European Union for type 1 diabetes mellitus) reduced the 2-NBDG phlorizin or LX4211 (Fig. 3e, Supplementary Fig. S4e) or shRNA uptake in H322/ER and HCC827/ER clones under low glucose (Fig. 3f and Supplementary Fig. S4f) also enhanced the glucose culture conditions (Fig. 2f and Supplementary Fig. S3i) without deprivation-induced caspase 3 or PARP cleavage and LC3β in affecting GLUT3 activity (Supplementary Fig. S3j). Similar suppres- H322/ER#2 and HCC827/ER#2 cells, but not in the parental H322 sive effects of SGLT1 shRNA on α-MDG uptake were also found in cells (Supplementary Fig. S4g). Conversely, overexpression of different H322/ER (Fig. 2g) and HCC827/ER (Supplementary Fig. SGLT1 was not only associated with higher α-MDG uptake S3k) clones. SGLT1 shRNA also reduced the glucose consumption (Supplementary Fig. S4h) and lower levels of apoptotic markers of H322/ER#1 cells (Supplementary Fig. S3l). These results suggest (Supplementary Fig. S4i) and cell death (Supplementary Fig. S4j) in that upregulation of SGLT1 mediates glucose uptake and thereby response to glucose deprivation in H322 cells, but SLGT1 supports the viability of acquired EGFR TKI-resistant cells. overexpression also attenuated erlotinib-induced PARP and caspase 3 cleavages in parental H322 cells (Fig. 3g) and HCC827 Targeting SGLT1 reduced EGFR TKI resistance in vitro and cells (Supplementary Fig. S4k) and consequently restored the TKI- in vivo suppressed viability (Fig. 3h and Supplementary Fig. S4l). These We next examined the role of SGLT1 in conferring drug resistance results support the contention that increased SGLT1 expression to EGFR TKIs through increasing glucose uptake. Treatments with enhances glucose uptake and thereby reduces the sensitivity of phlorizin or LX4211 decreased the proliferation rate of ER clones of NSCLC cells to EGFR TKIs.

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Fig. 3 The upregulated SGLT1 supported the cell viability of the acquired TKI-resistant cells. a The cell proliferation of H322/ER clones in response to erlotinib, phlorizin, or LX4211 was determined in WST-1 analysis. b The effects of phlorizin or LX4211 on cell viability of H322/ER clones under low glucose concentration were measured in WST-1 analysis. c, d The effects of SGLT1 shRNA on cell proliferation (c) and viability (d) of H322/ER clones under low glucose concentration were determined by cell counting and WST-1 analyses, respectively. e, f The effects of SGLT1 inhibitors (e) and shRNA (f) on caspase 3 or PARP cleavages or LC3β in H322/ER clones were analyzed by WB. g, h The effects of SGLT1 overexpression on the erlotinib-induced PARP and caspase 3 cleavages (g) and cell death (h) in H322 cells were examined in WB and WST-1 analyses, respectively. Data in (a)–(d), and (h) represent mean and sd from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 vs. control, Student’s t-test. Data in (e)–(g) are representative of three experiments.

Next, we addressed whether SGLT1 inhibitors can enhance the induced to mediate autophagy under glucose deprivation [44], in therapeutic efficacy of EGFR TKIs in tumor xenograft mouse the xenograft tumor tissues (Fig. 4b and Supplementary Fig. S5b). models. First, SCID mice bearing H292 subcutaneous xenografts To examine whether SGLT1 also plays a role in conferring intrinsic were treated with erlotinib (50 mg/kg), phlorizin (20 mg/kg), resistance to EGFR TKIs, the experimental metastatic NSCLC LX4211 (60 mg/kg) alone, or the combination of erlotinib with xenograft model was established by tail vein injection of SGLT1- either phlorizin or LX4211. Treatments with erlotinib, phlorizin, or positive and TKI-insensitive A549 cells transfected with the LX4211 alone did not significantly reduce tumor growth, but the luciferase gene. Co-treatment with erlotinib and phlorizin reduced combination of erlotinib with either phlorizin (Fig. 4a) or LX4211 the tumor size (Fig. 4c) and the number of tumor nodules (Fig. 4d). (Supplementary Fig. S5a) significantly suppressed the tumor Remarkable reductions in Ki67 expression, as well as increases in growth. After treatment with erlotinib for 29 days, levels of EGFR ATG7 expression and PARP cleavage, were also found in the lung and SGLT1 expression were elevated in the xenograft tumor tumor tissues after co-treatment with erlotinib and phlorizin (Fig. tissues in IHC staining analysis. The combination treatments also 4e). Together, these findings support the therapeutic potential of enhanced the reduction of the proliferation marker Ki67 and the SGLT1 inhibitors for reducing the development of acquired EGFR induction of apoptosis marker PARP cleavage and ATG7, which is TKI resistance in NSCLC patients.

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Fig. 4 Targeting SGLT1 reduced the development of acquired resistance to EGFR TKI in vivo. a The growth rate of xenograft tumors of H292 cells in response to treatments with erlotinib, phlorizin, or the combination was determined by measuring tumor size. b The representative IHC staining of tumor sections from (a) were shown (left) and the results from ﬁve independent sections for all groups were quantitated with H-score (Right). Scale bar, 50 μm. c–e SCID mice injected with A549-Luc cells were treated with erlotinib, phlorizin, or the combination. The tumor volumes were measured by detecting luminescent signals in the lumina LT in vivo imaging system (upper in panel (c)) and the luciferase activity was quantitated (lower in panel (c)). After treatment for 3 weeks, the lungs of A549 cell-xenograft SCID mice were harvested and the numbers of lung tumor nodules were quantitated. e The representative IHC staining of tumor sections from (d) was shown (left) and the results from ﬁve independent sections for all groups were quantitated with H-score (Right). Scale bar, 50 μm. Data represent mean ± sd *p < 0.05 and **p < 0.01 vs. control, Student’s t-test.

Oncogene (2021) 40:4796 – 4808 C.-H. Chen et al. 4802 SGLT1 is upregulated in recurrent tumor tissues after the cetuximab-induced cell death (Fig. 6i and Supplementary Fig. failure of EGFR TKI treatment and is associated with a poor S7h), PARP and caspase 3 cleavage, and AMPK activation (Fig. 6j) prognosis of NSCLC in parental NSCLC cells. These results support the notion that In the Kaplan–Meier survival analysis of records from the public EGFR, in the presence of TKIs, upregulates SGLT1 expression to transcriptomic database [45], higher SGLT1 mRNA expression support cell growth and survival in cells with acquired EGFR TKI correlated with an increasingly worse overall survival rate in lung resistance. adenocarcinoma patients (Supplementary Fig. S6a), which was reflected in both smoker and non-smoker populations (Supple- Phosphorylation of EGFR T678 by PKCδ mediates SGLT1 mentary Fig. S6b, c). We next analyzed the clinical correlation of protein stabilization by enhancing the interaction between SGLT1 expression with prognosis in 72 NSCLC patients who EGFR and SGLT1 carried either wt or mutant EGFR and received erlotinib or We next investigated how EGFR stabilizes SGLT1 in TKI-resistant gefitinib treatments. SGLT1 expression was associated with cells independent of its tyrosine kinase activity. Despite tyrosine gender and age, but not smoking behavior, EGFR mutation autophosphorylation, serine or threonine phosphorylation in status, tumor size, lymph node metastasis, pathological stage, or members of the ErbB family has been found to regulate their immediate response to EGFR TKIs, with higher SGLT1 expression, kinase activity, protein stability, or endocytosis [47, 48]. Interest- detected in NSCLC tumors of males aged over 55 years ingly, we observed that EGFR phosphorylations at T678 and (Supplementary Table S1 and Supplementary Fig. S6d, e). EGFR Ser1046/1047, but not T669, all of which are mediated by different TKI-treated NSCLC patients with the higher SGLT1-expressing Ser/Thr kinases [49–52], were increased in H322/ER clones while tumors showed a lower overall survival rate with statistical Y1068 phosphorylation was suppressed by erlotinib (Fig. 7a and significance (Fig. 5a) and a more unfavorable progression-free Supplementary Fig. S8a). The upregulation of EGFR T678 survival rate with marginal significance (Fig. 5b). The negative phosphorylation was also observed in the xenograft tumor tissue correlations of SGLT1 expression with overall and progression- of parental H292 cells after 1 month of erlotinib treatment (Fig. free survival rates were also observed in both NSCLC subpopula- 7b), in the xenograft tissues of H292/ER clones (Supplementary tions; in patients with wt EGFR tumors (Fig. 5c, d) and those with Fig. S8b), and in human NSCLC tumors with acquired TKI EGFR mutations (Fig. 5e, f), respectively. resistance (Fig. 7c), compared with their parental counterparts. We then examined the change in SGLT1 expression in human Mutation of T678, but not S1046/1047, to Ala abolished EGFR- NSCLC tumor tissues after the development of acquired resistance enhanced SGLT1 expression, and this effect was reversed by to EGFR TKIs. Treatment-naïve primary tumor tissues paired with treatment with proteasome inhibitor MG132 in HEK-293T cells the acquired EGFR TKI-resistant tumor tissues were collected from (Fig. 7d). Mutation of T678A also reduced the interaction between 9 NSCLC patients and were subjected to IHC staining with an anti- EGFR and SGLT1 (Fig. 7e). These results suggest that increased SGLT1 antibody. In response to acquired EGFR TKI resistance, T678 phosphorylation of EGFR mediates SGLT1 protein stabiliza- recurrent tumor tissues from 6 of 9 patients demonstrated tion through protein–protein interaction. upregulated SGLT1 expression (Fig. 5g). Interestingly, SGLT1 PKC has been reported to phosphorylate EGFR at T678 to expression was increased in all 5 male patients but was decreased protect EGFR from degradation [49, 50]. Protein kinase Cδ has in 3 of the 4 female patients (Fig. 5h). These results suggest that been found to contribute to the acquired resistance of NSCLCs SGLT1 upregulation may contribute to the acquired resistance to to EGFR TKIs [12], but the underlying mechanisms remain EGFR TKIs in NSCLC patients, especially in males. It is not yet clear unclear. Therefore, we next asked whether PKCδ is involved in whether hormone receptors are involved in the regulation of SGLT1-mediated TKI resistance through EGFR T678 phosphor- SGLT1 expression in lung cancer tissues and would be worthy of ylation. The viabilities of H322/ER#2 (Fig. 7f) and HCC827/ER#2 further investigation. (Supplementary Fig. S8c) cells were decreased by treatment with the pan-PKC inhibitors GO6983, staurosporine, and sotrasturin, Elevated EGFR expression stabilizes SGLT1 expression to in a dose-dependent manner. GO6983 also suppressed basal support the survival of TKI-resistant cells and glucose-induced ECAR (Supplementary Fig. S8d, e) and EGFR reportedly stabilizes SGLT1 expression, independent of its glucose uptake (Fig. 7g and Supplementary Fig. S8f) in H322/ tyrosine kinase activity [46]. We observed elevations in EGFR ER#2 and HCC827/ER#2 cells. These PKC inhibitors also expression after treatment with erlotinib (Fig. 4b and Supplemen- suppressed EGFR T678 phosphorylation and EGFR and SGLT1 tary Fig. S5b). Thus, we sought to determine whether the protein levels (Fig. 7h and Supplementary Fig. S8g). Over- upregulation of SGLT1 in TKI-resistant cells depends upon EGFR. expression of PKCδ induced increases in EGFR levels, EGFR T678 We observed increases in both SGLT1 expression and EGFR phosphorylation, and SGLT1 protein levels in H322 cells protein levels, but also a decrease in EGFR Y1068 phosphorylation, (Supplementary Fig. S8h), and further enhanced EGFR- a marker for EGFR tyrosine kinase activity, in the TKI-resistant H322 mediated SGLT1 upregulation, which was abolished by the clones in the presence of erlotinib (Fig. 6a). Similarly, EGFR mutation of EGFR T678A (Fig. 7i). These findings indicate that upregulation was also observed in the H292/ER xenograft tumor PKCδ, in addition to its nuclear functions [12], confers EGFR TKI tissues compared to that of their parental cells in SCID mice resistance by regulating the formation of the EGFR/SGLT1 (Supplementary Fig. S7a). Silencing EGFR expression with specific complex in the cytoplasm. siRNAs reduced the uptake of α-MDG (Fig. 6b, c) and 2-NBDG Interestingly, PKCδ S643/676 phosphorylation, which is (Supplementary Fig. S7b) in ER clones. Similarly, downregulation mediated by mTORC2, but not T505 phosphorylation, was of EGFR by treatment with cetuximab, the EGFR monoclonal observed in the H322/ER and HCC827/ER clones (Fig. 7j and antibody, suppressed α-MDG (Fig. 6d) and 2-NBDG (Supplemen- Supplementary Fig. S8i) and in the xenograft tumor tissues of tary Fig. S7c) uptake in ER clones. Treatments with EGFR siRNAs parental H292 cells after 1 month of erlotinib treatment (Fig. 7k). (Fig. 6e and Supplementary Fig. S7d) or cetuximab (Fig. 6f and Since mTORC2 is inactivated by S6K in response to the signaling of Supplementary Fig. S7e) also reduced the cell growth of H322/ER the EGFR/mTORC1 axis [53], the mTORC2/PKCδ axis may be and HCC827/ER clones in colony formation assays. Moreover, activated and mediate EGFR T678 phosphorylation for SGLT1 downregulation of EGFR by siRNA administration (Fig. 6g and protein stabilization, when EGFR tyrosine kinase activity was Supplementary Fig. S7f) or cetuximab (Fig. 6h and Supplementary suppressed by TKIs. In support of this notion, the mTORC1/2 Fig. S7g) reduced both EGFR and SGLT1 protein levels and also inhibitor everolimus suppressed not only PKCδ S643/676 phos- increased PARP and caspase 3 cleavages in H322/ER and HCC827/ phorylation, but also EGFR T678 phosphorylation and SGLT1 levels ER clones. In contrast, overexpression of SGLT1 attenuated (Supplementary Fig. S8j).

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Fig. 5 SGLT1 expression negatively correlates with the clinical beneﬁts of EGFR TKI in NSCLC patients. a, b Tumor tissues from total NSCLC patients ever treated with EGFR TKIs were subjected to IHC staining with anti-SGLT1 antibody ((a) upper). Scale bar, 50 μm. The clinical correlation of SGLT1 expression with overall survival (a) and progression-free survival (PFS) (b) rates were analyzed in Kaplan–Meier analysis. c– f The EGFR TKI-treated NSCLC patients in (a and b) were further classiﬁed into wt EGFR (c) and (d) and mutant EGFR (e) and (f) groups for Kaplan–Meier overall survival and progression-free survival analysis. g, h SGLT1 protein levels in the paired tissues from treatment-naïve tumors and acquired TKI-resistant tumors of nine lung cancer patients were examined by IHC staining (upper in panel (g)) and quantitated (lower in panel (g)). The results from panel (g) were further divided into two groups according to the genders of patients (h). Scale bar, 50 μm.

In conclusion, TKIs suppressed EGFR and its downstream Akt increase in the intracellular AMP/ATP ratio, and activation of AMPK and ERK signaling pathways, as well as the membrane levels of for the suppression of mTORC1 and tumor growth (Fig. 8, left). GLUT and glucose uptake in the sensitive cells. Reduced glucose However, while EGFR tyrosine kinase activity was suppressed by uptake led to a subsequent reduction in ATP production, an long-term TKI treatment, mTORC2 was activated from the EGFR/

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Fig. 6 The increased EGFR mediated glucose uptake and viability of the acquired erlotinib-resistant cells through SGLT1 upregulation. a The protein expressions of the ErbB family and SGLT1 in H322 cells and their ER clones were analyzed in WB analysis. b–h The effects of EGFR siRNA or monoclonal antibody cetuximab on α-MDG uptake (b)–(d), colony formation (e) and (f), and caspase and PARP cleavages (g) and (h) of H322/ER#2 and HCC827/ER#2 cells were examined, respectively. i, j The effects of SGLT1 overexpression on the cetuximab-induced viability inhibition (i) or PARP and caspase 3 cleavages (j) of H322 cells were examined in WST-1 analysis and WB, respectively. Data in (c) and (d) and (i) represent mean ± sd from three independent experiments. *p < 0.05; ***p < 0.001 vs. control, Student’s t-test. Data in (a), (e)–(h), and (j) are representative of three experiments.

mTORC1/S6K axis to phosphorylate PKCδ for subsequent EGFR and lactate production by inhibiting the translocation of the Glut3 T678 phosphorylation and SGLT1 protein stabilization. Even in an transporter from the cytosol to the plasma membrane in lung environment of low glucose, higher SGLT1 expression engulfs adenocarcinoma [24]. The downregulation of Glut1 and Glut3 more glucose to maintain intracellular glucose levels, leading to protein levels also accounts for the anticancer activity of EGFR TKIs acquired EGFR TKI resistance and maintenance of ATP production in NSCLC cell lines and xenograft tumor tissues [24, 25]. EGFR for cell viability (Fig. 8, right). Thus, co-treatment with SGLT1 expression and downstream signaling pathways have shown inhibitors may avoid the development of acquired resistance to positive correlations with Glut1 expression and membrane EGFR TKIs. localization in lung cancer [54], pancreatic cancer tissue [55, 56], and triple-negative breast cancer (TNBC) [57], suggesting that TKI- sensitive cancer cells employ passive glucose transporters to DISCUSSION engulf glucose and that downregulation of these transporters may In addition to the inhibition of EGFR downstream PI3K/Akt and account for the anticancer activity of TKIs. Under adaptation to MAPK survival pathways, reductions in glucose uptake and long-term TKI treatment, however, SGLT1 upregulation compen- glycolysis have been detected in geﬁtinib-treated lung cancer sated for glucose uptake in TKI-resistant cells in our study. cells that precede cell cycle suppression and apoptosis induction SGLT1 is generally expressed in the normal epithelial cells of the [22], suggesting that glucose metabolic activity closely reﬂects the small intestine to transport glucose and galactose across the intrinsic response to EGFR TKI-based therapy. However, it remains luminal side of enterocytes, but its overexpression in prostate unclear as to whether the development of acquired resistance to cancer [58] and colorectal cancer [59] is associated with poor EGFR TKIs involves the reprogramming of glucose metabolism in prognosis. Although SGLT2, typically expressed in the renal NSCLC cells. In this study, our results showed that protein stability proximal tubule, has also been reported to play a critical role in of the active glucose transporter SGLT1 was dramatically the development of pancreatic or breast cancer [60, 61], our data enhanced by EGFR relying on PKCδ-mediated phosphorylation showed that SGLT1, but not SGLT2, is upregulated in response to to support glucose uptake and viability of ER lung cancer cells. EGFR TKI treatment, which supports glucose uptake and cellular Cancer cells support their growth by expressing glucose viability in NSCLC cells with acquired TKI resistance. Although the transporters that increase glucose uptake from the extracellular EGF-activated PI3K/Akt/CREB signaling axis upregulates SGLT1 environment. Treatment with TKIs decreases glucose consumption expression and enhances glucose uptake in intestine epithelial

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Fig. 7 EGFR Thr678 phosphorylation by PKC delta mediated the SGLT1/EGFR interaction for SGLT1 protein stabilization. a EGFR phosphorylations in H322 cells and their ER clones were examined in WB with the indicated antibodies. b EGFR pT678 phosphorylation in the expression of IHC pictures in lung sections (left) and the H-score of protein expression was shown (right). Scale bar, 50 μm. c EGFR T678 phosphorylation and EGFR protein level in the paired treatment-naïve and acquired TKI-resistant tumor tissues of lung cancer patients was determined by IHC staining. Scale bar, 50 μm. d, e The indicated protein expression in HEK-293T cells co-transfected with EGFR mutants and SGLT1 in the presence or absence of MG-132 was analyzed by WB (d) and IP/WB (e). f The effects of GO6983, staurosporine, or sotrasturin on cell viability were determined in WST-1 assay. g Glucose uptake in H322/ER cells in response to GO6983 treatment was analyzed with the 2- NBDG uptake assay. Scale bar, 330 μm. h–j The total lysates from H322/ER#2 cells treated with various PKC inhibitors (h), HEK-293T cells co- transfected with EGFR mutants, and PKCδ (i), and H322 cells and ER clones (h) were subjected to WB with the indicated antibodies. k PKCδ pS643/676 and PKCδ expression in the H292 cells-xenograft tumor tissues in response to erlotinib treatment were examined in IHC analysis (left panel), and the H-score of protein expression were shown (right). Scale bar, 50 μm. Data are shown in (a) and (d)–(j) represent mean and s. d. from three experiments. Data in (b) and (k) are representative of ﬁve experiments and shown as mean ± sd. *p < 0.05; **p < 0.01; ***p < 0.001 vs. control group, Student’s t-test.

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Fig. 8 A proposed model illustrating the involvement of SGLT1-mediated metabolic reprogramming in the development of acquired resistance to EGFR TKIs. In sensitive NSCLC cells, EGFR TKIs suppressed EGFR and its downstream Akt and ERK signaling pathways as well as the membrane level of GLUT and glucose uptake. Reduced glucose uptake leads to lower ATP production, an increase in the intracellular AMP/ ATP ratio, and activation of AMPK for the suppression of mTORC1 and tumor growth. In the presence of TKIs, however, PKCδ is activated by mTORC2 to phosphorylate EGFR at T678 to stabilize SGLT1 protein in an EGFR tyrosine kinase-independent manner while EGFR/mTORC1/S6K axis was inhibited. Even under a low glucose environment, the elevated SGLT1 engulfs more glucose to maintain intracellular glucose level and subsequently ATP production for cell survival, leading to the acquired EGFR TKI resistance. Co-treatment with SGLT1 inhibitors may avoid the development of acquired EGFR TKI resistance.

cells [62], the tyrosine kinase activity of EGFR or IGF1R and Akt emerged as a possibility [73]. Four SGLT2 inhibitors, empagliflozin signals are not needed for SGLT1 upregulation in cancer cells (Jardiance), dapagliflozin (Forxiga), canagliflozin (Invokana), and [46, 63]. In this study, our data showed that enhanced SGLT1 ertugliflozin (Stelagro) have recently been approved for the protein expression and glucose uptake in TKI-resistant cells were treatment of type 2 diabetes mellitus by the US Food and Drug mediated by kinase-inactive EGFR (Fig. 6). Downregulation of EGFR Administration, and are associated with beneficial effects in the expression involves the direct targeting of its 3′UTR activity by cardiovascular system and the kidney [74]. Randomized clinical microRNA-7 [64–67]. Interestingly, the kinase-dependent function- trial data have not revealed any associations between treatment ing of EGFR has been reported to induce miR-7 transcription [68], with these SGLT2 inhibitors and increased incidence rates of suggesting that miR-7 acts as a negative feedback regulator of malignancies [75]. Our data show that co-targeting SGLT1 with EGFR expression. Indeed, EGFR expression without tyrosine kinase LX4211 (sotagliflozin) enhanced the anticancer activity of EGFR activity is increased in response to TKI treatment, which down- TKIs in mouse models. LX4211 is an orally-delivered dual SGLT1/2 regulates microRNA-7 and promotes the migration and invasion of inhibitor approved for T1DM and exhibits a 20-fold higher potency TNBC cells [64, 69]. This negative feedback regulation of EGFR may for SGLT2 over SGLT1 [76]; the involvement of SGLT2 inhibition in also contribute to the protein stabilization of SGLT1 in NSCLC cells this synergism cannot be ruled out. Moreover, this dual inhibitor with acquired EGFR TKI resistance. improves severe glycemic and non-glycemic outcomes without Besides the elevation of EGFR protein levels, our data revealed that severe gastrointestinal side effects [77], although its associated PKCδ-mediated EGFR T678 phosphorylation also enhanced the increased risk of diabetic ketoacidosis in patients with type 1 protein–protein interaction of EGFR with SGLT1 to stabilize SGLT1 diabetes mellitus is of concern [78]. The possibility of this event protein levels in TKI-resistant cells (Fig. 7). This conserved phosphor- should be evaluated during clinical testing of the synergistic ylation by PKC reduces endocytic trafficking of EGFR and ErbB3 effects of LX4211 on the anticancer activity of EGFR TKIs. [70, 71], and the retention of these receptors on the plasma In conclusion, our results elucidate the role of PKCδ/EGFR- membrane may account for the higher interaction between EGFR dependent SGLT1 expression in the rewiring of glucose uptake and SGLT1 in TKI-resistant cells. Moreover, our data found that and metabolism that supports the expansion of cells with mTORC2-mediated PKCδ phosphorylation (Ser643 and Ser676) was acquired TKI resistance (Fig. 8). These findings indicate that SGLT1 notably increased in the cells with acquired resistance to erlotinib is a potential target for avoiding acquired resistance to EGFR TKI and that mTORC1/2 inhibition suppressed PKCδ activation and EGFR/ therapy in NSCLC. SGLT1 protein expression. PKCδ-mediated EGFR/SGLT1 stabilization may be involved in the mTORC2-mediated metabolic reprogramming in EGFR TKI-resistant NSCLC cells [72]. These findings also AVAILABILITY OF MATERIALS AND METHODS support targeting of mTORC2, which is activated due to the The materials and methods used in this study are available in the Supporting inhibition of EGFR/mTORC1/S6K signaling [53], as another therapeu- Information. tic strategy to overcome EGFR TKI resistance in NSCLC cells [72]. Since SGLTs are functionally active in various cancer types, REFERENCES including pancreatic, prostate, and brain cancers [60], the use of 1. Skoulidis F, Heymach JV. Co-occurring genomic alterations in non-small-cell lung new antidiabetic SGLT inhibitors for cancer therapy has also cancer biology and therapy. Nat Rev Cancer. 2019;19:495–509.

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Resistance to the tyrosine kinase Research Core Facilities in the Office of Research & Development at China Medical inhibitor axitinib is associated with increased glucose metabolism in pancreatic University, Taichung, Taiwan. This research was funded in part by the following: adenocarcinoma. Cell Death Dis. 2014;5:e1160. Ministry of Science Technology, Taiwan (grant number: MOST 108–2314-B-039–032), 57. Hussein YR, Bandyopadhyay S, Semaan A, Ahmed Q, Albashiti B, Jazaerly T, et al. China Medical University (grant number: CMU106-ASIA-18), and China Medical Glut-1 expression correlates with basal-like breast cancer. Transl Oncol. 2011;4:321–7. University Hospital (DMR-108–012, DMR-109–212, DMR-109–213). This work was also 58. Ren J, Bollu LR, Su F, Gao G, Xu L, Huang WC, et al. EGFR-SGLT1 interaction does financially supported by the “Drug Development Center, China Medical University” not respond to EGFR modulators, but inhibition of SGLT1 sensitizes prostate from The Featured Areas Research Center Program within the framework of the cancer cells to EGFR tyrosine kinase inhibitors. Prostate. 2013;73:1453–61. Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. Y. 59. Guo GF, Cai YC, Zhang B, Xu RH, Qiu HJ, Xia LP, et al. Overexpression of SGLT1 Wang was supported by Medical Research Council (MR/S025480/1). We would like to and EGFR in colorectal cancer showing a correlation with the prognosis. Med thank Iona J. MacDonald from China Medical University, Taichung, Taiwan, for her Oncol. 2011;28(Suppl 1):S197–203. English language revision of this manuscript. 60. Scafoglio C, Hirayama BA, Kepe V, Liu J, Ghezzi C, Satyamurthy N, et al. Functional expression of sodium-glucose transporters in cancer. 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A kinase-independent biological samples, performed the experiments, and provided clinical information. W-CH activity for insulin growth factor-1 receptor (IGF-1R): implications for inhibition of supervised the entire project and wrote the manuscript. M-CH provided scientific the IGF-1R signal. Oncotarget. 2013;4:463–73. input and wrote the manuscript. 64. Hsia TC, Tu CY, Chen YJ, Wei YL, Yu MC, Hsu SC, et al. Lapatinib-mediated cyclooxygenase-2 expression via epidermal growth factor receptor/HuR interaction enhances the aggressiveness of triple-negative breast cancer cells. Mol Compliance with ethical standards Pharmacol. 2013;83:857–69. 65. Tu CY, Chen CH, Hsia TC, Hsu MH, Wei YL, Yu MC, et al. Trichostatin A suppresses EGFR expression through induction of microRNA-7 in an HDAC-independent CONFLICT OF INTEREST manner in lapatinib-treated cells. BioMed Res Int. 2014;2014:168949. The authors declare no competing interests. 66. 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Eur J Pharm. 2018;822:25–31. Attribution 4.0 International License, which permits use, sharing, 74. Rieg T, Vallon V. Development of SGLT1 and SGLT2 inhibitors. Diabetologia. adaptation, distribution and reproduction in any medium or format, as long as you give 2018;61:2079–86. appropriate credit to the original author(s) and the source, provide a link to the Creative 75. Dicembrini I, Nreu B, Mannucci E, Monami M. Sodium-glucose co-transporter-2 Commons license, and indicate if changes were made. The images or other third party (SGLT-2) inhibitors and cancer: A meta-analysis of randomized controlled trials. material in this article are included in the article’s Creative Commons license, unless Diabetes Obes Metab. 2019;21:1871–7. indicated otherwise in a credit line to the material. If material is not included in the 76. Lapuerta P, Zambrowicz B, Strumph P, Sands A. Development of sotagliflozin, a article’s Creative Commons license and your intended use is not permitted by statutory dual sodium-dependent glucose transporter 1/2 inhibitor. Diab Vasc Dis Res. regulation or exceeds the permitted use, you will need to obtain permission directly 2015;12:101–10. from the copyright holder. To view a copy of this license, visit http://creativecommons. 77. Zambrowicz B, Freiman J, Brown PM, Frazier KS, Turnage A, Bronner J, et al. org/licenses/by/4.0/. LX4211, a dual SGLT1/SGLT2 inhibitor, improved glycemic control in patients with type 2 diabetes in a randomized, placebo-controlled trial. Clin Pharm Ther. 2012;92:158–69. © The Author(s) 2021

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